(1) Field of the Invention
The present invention relates to a filler for measuring the enzyme activity, an enzyme activity-measuring column packed with the filler, and a method of measuring the enzyme activity by using the column.
(2) Description of the Related Art
It is known that an enzyme is a protein synthesized by living cells, and the catalytic action of the enzyme to a living body reaction is important for the maintenance of life. In the natural state, millions of enzymes originating in animals, plants and micro-organisms exist, and about 2,000 of these enzymes have been isolated and are used as industrial enzymes in the fields of foodstuffs and detergents or as medicinal enzymes for remedy of diseases and clinical examination, and recently, in the field of genetic engineering.
The measurement of the activity of an enzyme is made by measuring the catalytic activity of the enzyme and obtain a basic index showing the function of the enzyme. In the conventional method, the enzyme activity is measured by analyzing the decrease in the amount of a substrate or the increase in the amount of a product. More specifically often adopted are a method in which the decrease in the amount of a substrate or the increase in the amount of a product after the reaction by an enzyme is measured as the absorbance by a spectrophotometer, a method in which the measurement is carried out by coloration of a substrate or product by a chemical reagent, and a method in which a product is converted to a coloring substance and the measurement is effected on the formed coloring substance. Moreover, a method is known in which a substrate is labelled with a radioactive element and the labelled substrate is detected. In general, however, these methods have problems in that a large quantity of the enzyme is necessary for the measurement, the chemical reagent used is expensive, many kinds of chemical reagents are necessary, a long time is required for the measurement, a high degree of skill is needed when making the measurement, and the measurement is easily influenced by impurities. Accordingly, it is difficult to obtain an accurate value.
As the means for coping with these defects, a method has been proposed in which the measurement is combined with liquid chromatography, i.e., an enzyme is separated and purified by a column and after elution from the column, the enzyme is placed in contact with a substrate solution, and the decrease in the amount of the substrate determined [H. A. Chase: J. Chem. Tech. Biotechnol., 36, 351 (1986), and N. Ito et al: J. Chromatogr., 400, 163 (1987)] this method is still unsatisfactory in that high-purity and expensive reagents must be continuously supplied and an additional device such as a pump must be used to ensure this continuous supply.